Recombinant neospora antigens and their uses

ABSTRACT

The present invention provides isolated bovine Neospora cultures. The cultures are used to develop diagnostic assays for the detection of Neospora infections in cattle and other animals. Also provided are pharmaceutical compositions for the treatment and prevention of Neospora infections.

BACKGROUND OF THE INVENTION

This invention relates to the diagnosis and prevention of bovinediseases caused by the protozoan parasite, Neospora. The inventionspecifically relates to isolated cultures of the parasite and nucleicacids and proteins isolated from them.

A distinct pattern of inflammatory lesions, consisting of focalnon-suppurative necrotizing encephalitis, non-suppurative myocarditisand myositis have been observed in many aborted bovine fetuses submittedfor diagnosis. The pattern of lesions, particularly in the brain, issimilar to those seen with Toxoplasma gondii infections in sheep.However, cattle have been reported to be resistant to T. gondiiinfection Dubey, Vet. Parasit. 22:177-202 (1986). In 1988, acyst-forming protozoal parasite was first identified byhistopathological examination in fetuses (Barr et al., Vet. Parasit.27:354-61 (1990)). This parasite was morphologically similar toToxoplasma, except that some of the cysts had thick walls, which wasmore similar to the Neospora caninum-like protozoan observed by Thilsted& Dubey (J. Vet. Diagnos. Invest. 1:205-9 (1989)) in aborted fetusesfrom a dairy in New Mexico.

Further studies showed that the protozoal parasites associated withinflammatory lesions in aborted fetuses and neonatal calves inCalifornia had ultrastructural and antigenic features that were mostsimilar to N. caninum parasites which were originally isolated from dogs(Dubey et al., JAVMA 193:1259-63 (1988)). However, differences in theantigenic reactivity of the bovine protozoan and N. caninum when testedwith a panel of antisera indicated that they may not be the same species(Barr et al., Vet. Pathol. 28:110-16 (1991)).

A more complete understanding of the identity and biology of thesebovine protozoa requires establishing continuous in vitro cultures ofthe parasites. Such cultures would also be valuable in the developmentof diagnostic assays and pharmaceutical compositions for the treatmentand prevention of Neospora infections. The present invention addressesthese and other needs.

SUMMARY OF THE INVENTION

The present invention provides biologically pure cultures of bovineNeospora. Two such cultures have been deposited with the ATCC and givenATCC Accession No. 75710 (BPA1), and ATCC Accession No. 75711 (BPA 6).

The invention also provides methods of detecting the presence ofantibodies specifically immunoreactive with a bovine Neospora antigen ina biological sample (e.g., bovine serum). The method comprisescontacting the sample with the Neospora antigen, thereby forming anantigen/antibody complex, and detecting the presence or absence of thecomplex. The Neospora antigen is typically an isolated recombinantlyproduced immunodominant Neospora antigen. In some embodiments, theantigen is immobilized on a solid surface and the complex is detectedusing a fluorescently labeled anti-bovine antibody.

The invention further provides methods of detecting the presence ofNeospora in a biological sample. These methods comprise contacting thesample with an antibody specifically immunoreactive with a Neosporaantigen, thereby forming an antigen/antibody complex, and detecting thepresence or absence of the complex. The antibody (e.g., a monoclonalantibody) may be immobilized on a solid surface and the complex detectedusing a second labeled antibody. Typically, the biological sample isbovine fetal neurological tissue.

The methods of the invention also include detecting the presence ofNeospora-specific nucleic acids in a biological sample by contacting thesample with an oligonucleotide probe which specifically hybridizes witha target Neospora-specific polynucleotide sequence and detecting thepresence or absence of hybridization complexes. The methods may furthercomprise amplifying the target Neospora-specific polynucleotidesequence.

The invention further provides for pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and an immunogenicallyeffective amount of a bovine Neospora antigen, such as a recombinantlyproduced bovine Neospora polypeptide.

The pharmnaceutical compositions are used in protecting a bovine animalfrom infection by bovine Neospora. The compositions are preferablyadministered to a cow or heifer when the animal is breeding. Thepharmaceutical composition is usually administered parenterally.

DEFINITIONS

"Antibody" refers to an immunoglobulin molecule able to bind to aspecific epitope on an antigen. Antibodies can be a polyclonal mixtureor monoclonal. Antibodies can be intact immunoglobulins derived fromnatural sources or from recombinant sources and can be immunoreactiveportions of intact immunoglobulins. Antibodies may exist in a variety offorms including, for example, Fv, F_(ab), and F(ab)₂, as well as insingle chains. Single-chain antibodies, in which genes for a heavy chainand a light chain are combined into a single coding sequence, may alsobe used.

"Biological sample" refers to any sample obtained from a living or deadorganism. Examples of biological samples include biological fluids andtissue specimens. Examples of tissue specimens include fetal braintissue, spinal cord, and placenta. Examples of biological fluids includeblood, serum, plasma, urine, ascites fluid, cerebrospinal fluid andfetal fluid.

A "biologically pure bovine Neospora culture" refers to a continuous invitro culture of bovine Neospora organisms (e.g. tachyzoites) which issubstantially free of other organisms other than the host cells in whichNeospora tachyzoites are grown. A culture is substantially free of otherorganisms if standard harvesting procedures (as described below) resultin a preparation which comprises at least about 95%, preferably 99% ormore of the organism, e.g., Neospora tachyzoites.

"Bovine Neospora" refers to Neospora or "Neospora-like" protozoansidentified in or isolated from bovine tissues and fluids. Typically, theprotozoal parasites can be isolated from neurological tissue of abortedbovine fetuses or congenitally infected calves. Exemplary isolates havebeen deposited with the American Type Culture Collection, as describedbelow.

A bovine Neospora "protein" or "polypeptide" includes allelic variationsnormally found in the natural population and changes introduced byrecombinant techniques. Those of skill recognize that proteins can bemodified in a variety of ways including the addition, deletion andsubstitution of amino acids.

A "recombinantly produced immunodominant Neospora antigen" is arecombinantly produced polypeptide comprising one or more immunodominantepitopes. Exemplary recombinant antigens of the invention include SEQ.ID. NO: 10 and SEQ ID NO: 12. Such antigens are encoded by SEQ. ID. NO:9 and SEQ ID NO: 11. Terms used to describe the recombinant antigens andthe nucleic acids which encode them will be understood by those of skillin the art to include sequences which are substantially identical to theexemplified sequences. Substantial identity can be determined asdescribed below.

"Nucleic acids" and "polynucleotides", as used herein, may be DNA orRNA. One of skill will recognize that for use in the expression ofNeospora proteins or as diagnostic probes, polynucleotide sequences neednot be identical and may be substantially identical to sequencesdisclosed here. In particular, where the inserted polynucleotidesequence is transcribed and translated to produce a functionalpolypeptide, one of skill will recognize that because of codondegeneracy a number of polynucleotide sequences will encode the samepolypeptide.

"Percentage of sequence identity" for polynucleotides and polypeptidesis determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Optimal alignment ofsequences for comparison may be conducted by computerizedimplementations of known algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by inspection.

Substantial identity of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 75% sequenceidentity, preferably at least 80%, more preferably at least 90% and mostpreferably at least 95%, Typically, two polypeptides are considered tobe substantially identical if at least 40%, preferably at least 60%,more preferably at least 90%, and most preferably at least 95% areidentical or conservative substitutions. Sequences are preferablycompared to a reference sequence using GAP using default parameters.

Another indication that polynucleotide sequences are substantiallyidentical is if two molecules hybridize to each other tinder stringentconditions. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Typicallystringent conditions for a Southern blot protocol involve washing atroom temperature with a 5× SSC, 0.1% SDS wash.

The phrase "selectively hybridizing to", refers to a hybridizationbetween a probe and a target sequence in which the probe bindssubstantially only to the target sequence when the target is in aheterogeneous mixture of polynucleotides and other compounds. Suchhybridization is determinative of the presence of the target sequence.Although the probe may bind other unrelated sequences, at least 90%,preferably 95% or more of the hybridization complexes formed are withthe target sequence.

The phrase "specifically immunoreactive with", when referring to aprotein or peptide, refers to a binding reaction between the protein andan antibody which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other compounds.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein and are described in detail below.

The phrase "substantially pure" or "isolated" when referring to aNeospora peptide or protein, means a chemical composition which is freeof other subcellular components of the Neospora organism. Typically, amonomeric protein is substantially pure when at least about 85% or moreof a sample exhibits a single polypeptide backbone. Minor variants orchemical modifications may typically share the same polypeptidesequence. Depending on the purification procedure, purities of 85%, andpreferably over 95% pure are possible. Protein purity or homogeneity maybe indicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band on a polyacrylamide gel uponsilver staining. For certain purposes high resolution will be needed andHPLC or a similar means for purification utilized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows indirect fluorescent antibody (IFA) titers of serialsamples from four cows that aborted Neospora-infected fetuses.

FIG. 2 shows IFA test titers of serial samples from two cows thataborted Neospora-infected fetuses and subsequently deliveredcongenitally infected calves.

FIG. 3 shows seroconversion by heifers experimentally infected withNeospora.

FIG. 4 shows IFA test titers: comparing cattle with no evidence ofinfection to dams with Neospora-infected fetuses or calves.

FIGS. 5A and 5B show sequence motifs in two immunodominant cDNA clonesisolated from Neospora (FIG. 5A, SEQ ID NO: 10; SPPQS(S/Y)PPEP=SEQ IDNO: 13; HP(H/Y)P=SEQ ID NO: 14; SPP(E/Q)=SEQ ID NO: 15;SY(A/P)P(D/E)PS=SEQ ID NO: 17; FIG. 5B, SEQ ID NO: 16).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides Neospora cultures isolated from cattle.The cultures are useful in a variety of applications including theproduction of nucleic acids or proteins for diagnostic assays and thepreparation of immunogenic proteins for use in vaccine compositions.

Neospora tachyzoite cultures of the invention have been deposited withthe American Type Culture Collection, Rockville, Md. on Mar. 17, 1994and given Accession Numbers 75710 (BPA1) and 75711 (BPA6).

These isolates were obtained from tissue homogenates of brain and/orspinal cord of an aborted bovine fetus and congenitally infected calves.Immunohistochemistry was used to identify the tachyzoite and/or cystsassociated with lesions in the tissues of these fetuses and calves asNeospora parasites prior to isolation. Tachyzoite stages of the isolateswere grown in stationary monolayer cultures of bovine fetaltrophoblasts, aortic endothelial cells and/or macrophages. Electronmicroscopic studies were used to characterize the ultrastructuralfeatures of the BPA1 isolate. Antigenically, tachyzoites of 5 separateisolates react strongly with antisera to Neospora and show little or noreactivity with antisera to Toxoplasma gondii or Hammondia hammondi.Based on these ultrastructural and antigenic characteristics, theseparasites can be distinguished from the most closely related andmorphologically similar genera of protozoa, Toxoplasma, Hammondia andSarcocystis.

In addition, partial sequences (500-550 base pairs) of the 5' end of thenuclear small subunit (nss)-rRNA gene for three of the bovine Neosporaisolates (BPA1, BPA3 and BPA5) have been obtained and shown to beidentical. The more complete 1.8 kilobase sequence of the nss-rRNA geneof the BPA1 isolate was obtained and compared to the sequences for thisgene in other coccidial parasites. Alignment of these sequences withpublished sequences of Neospora caninum, Cryptosporidium parvum,Sarcocystis muris and Toxoplasma gondii showed that the bovine Neosporaisolate is genotypically unique.

As explained in detail below, the isolates are used to develop a varietyof diagnostic assays as well as pharmaceutical compositions fortreatment and prevention of infection.

Preparation of Neospora Polypeptides and Nucleic Acids

Standard protein purification techniques can be used to isolate proteinsfrom the tachyzoites or bradyzoites derived from the cultures providedhere. Such techniques include selective precipitation with suchsubstances as ammonium sulfate, column chromatography,immunopurification methods, and the like. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982).

Using standard immunoblot techniques 11 proteins with molecular weightsof approximately 106, 49.5, 48, 33, 32.5, 30, 28, 26, 19, 18 and 17kilodaltons (kd) have been identified. All of these proteins arespecifically recognized by antibodies from Neospora infected cattle.Standard protein purification methods can be used to purify theseproteins and produce polyclonal or monoclonal antibodies for use in thediagnostic methods described below. Two of these antigens (approximately106 and 19 kd) have been shown to be useful in enzyme-linkedimmunoassays (ELISA) for the detection of Neospora-specific antibodiesin infected cattle.

Rather than extract the proteins directly from cultured tachyzoites,nucleic acids derived from the cultures can be used for recombinantexpression of immunodominant antigens of the invention. In thesemethods, the nucleic acids encoding the proteins of interest areintroduced into suitable host cells, followed by induction of the cellsto produce large amounts of the protein. The isolation of two exemplarynucleic acids is described in Example 6, below. The invention relies onroutine techniques in the field of recombinant genetics, well known tothose of ordinary skill in the art. A basic text disclosing the generalmethods of use in this invention is Sambrook et al., Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Publish., Cold Spring Harbor,N.Y. 2nd ed. (1989).

Nucleic acids for use as diagnostic oligonucleotide probes or for therecombinant expression of proteins can be isolated using a number oftechniques. For instance, portions of proteins isolated from thecultures discussed above can be sequenced and used to design degenerateoligonucleotide probes to screen a cDNA library. Amino acid sequencingis performed and oligonucleotide probes are synthesized according tostandard techniques as described, for instance, in Sambrook et al.,supra. Alternatively, oligonucleotide probes useful for identificationof desired genes can also be prepared from conserved regions of relatedgenes in other species.

Alternatively, polymerase chain reaction technology (PCR) can be used toamplify nucleic acid sequences of the desired gene directly from mRNA,from cDNA, from genomic libraries or cDNA libraries. Polymerase chainreaction (PCR) or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the mRNA in physiological samples, for nucleicacid sequencing, or for other purposes. For a general overview of PCRsee PCR Protocols: A Guide to Methods and Applications. (Innis, M,Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego(1990).

Standard transfection methods are used to produce prokaryotic,mammalian, yeast or insect cell lines which express large quantities ofthe desired polypeptide, which is then purified using standardtechniques. See, e.g., Colley et al., J. Biol. Chem. 264:17619-17622,1989; and Guide to Protein Purification, supra.

The nucleotide sequences used to transfect the host cells can bemodified to yield Neospora polypeptides with a variety of desiredproperties. For example, the polypeptides can vary from thenaturally-occurring sequence at the primary structure level by aminoacid, insertions, substitutions, deletions, and the like. Thesemodifications can be used in a number of combinations to produce thefinal modified protein chain.

The amino acid sequence variants can be prepared with various objectivesin mind, including facilitating purification and preparation of therecombinant polypeptide. The modified polypeptides are also useful formodifying plasma half life, improving therapeutic efficacy, andlessening the severity or occurrence of side effects during therapeuticuse. The amino acid sequence variants are usually predetermined variantsnot found in nature but exhibit the same immunogenic activity asnaturally occurring protein. In general, modifications of the sequencesencoding the polypeptides may be readily accomplished by a variety ofwell-known techniques, such as site-directed mutagenesis (see, Gillmanand Smith, Gene 8:81-97, 1979) and Roberts, S. et al., Nature328:731-734, 1987). One of ordinary skill will appreciate that theeffect of many mutations is difficult to predict. Thus, mostmodifications are evaluated by routine screening in a suitable assay forthe desired characteristic. For instance, the effect of variousmodifications on the ability of the polypeptide to elicit a protectiveimmune response can be easily determined using in vitro assays. Forinstance, the polypeptides can be tested for their ability to inducelymphoproliferation, T cell cytotoxicity, or cytokine production usingstandard techniques.

The particular procedure used to introduce the genetic material into thehost cell for expression of the polypeptide is not particularlycritical. Any of the well known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, spheroplasts, electroporation,liposomes, microinjection, plasmid vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (seeSambrook et al., supra). It is only necessary that the particularprocedure utilized be capable of successfully introducing at least onegene into the host cell which is capable of expressing the gene.

Any of a number of well known cells and cell lines can be used toexpress the polypeptides of the invention. For instance, prokaryoticcells such as E. coli can be used. Eukaryotic cells include, yeast,Chinese hamster ovary (CHO) cells, COS cells, and insect cells.

The particular vector used to transport the genetic information into thecell is also not particularly critical. Any of the conventional vectorsused for expression of recombinant proteins in prokaryotic andeukaryotic cells may be used. Expression vectors for mammalian cellstypically contain regulatory elements from eukaryotic viruses.

The expression vector typically contains a transcription unit orexpression cassette that contains all the elements required for theexpression of the polypeptide DNA in the host cells. A typicalexpression cassette contains a promoter operably linked to the DNAsequence encoding a polypeptide and signals required for efficientpolyadenylation of the transcript. The term "operably linked" as usedherein refers to linkage of a promoter upstream from a DNA sequence suchthat the promoter mediates transcription of the DNA sequence. Thepromoter is preferably positioned about the same distance from theheterologous transcription start site as it is from the transcriptionstart site in its natural setting. As is known in the art, however, somevariation in this distance can be accommodated without loss of promoterfunction.

Following the growth of the recombinant cells and expression of thepolypeptide, the culture medium is harvested for purification of thesecreted protein. The media are typically clarified by centrifugation orfiltration to remove cells and cell debris and the proteins areconcentrated by adsorption to any suitable resin or by use of ammoniumsulfate fractionation, polyethylene glycol precipitation, or byultrafiltration. Other routine means known in the art may be equallysuitable. Further purification of the polypeptide can be accomplished bystandard techniques, for example, affinity chromatography, ion exchangechromatography, sizing chromatography, His₆ tagging and Ni-agarosechromatography (as described in Dobeli et al. Mol. and Biochem. Parasit.41:259-268 (1990)), or other protein purification techniques to obtainhomogeneity. The purified proteins are then used to producepharmaceutical compositions, as described below.

An alternative method of preparing recombinant polypeptides useful asvaccines involves the use of recombinant viruses (e.g., vaccinia).Vaccinia virus is grown in suitable cultured mammalian cells such as theHeLa S3 spinner cells, as described by Mackett, Smith and Moss, "Theconstruction and characterization of Vaccinia Virus RecombinantsExpressing Foreign Genes" in "DNA cloning Vol. II. A practicalapproach", Ed. D. M. Glover, IRL Press, Oxford, pp 191-211.

Antibody Production

The isolated proteins or cultures of the present invention can be usedto produce antibodies specifically reactive with Neospora antigens. Ifisolated proteins are used, they may be recombinantly produced orisolated from Neospora cultures. Synthetic peptides made using theprotein sequences may also be used.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. When appropriatelyhigh titers of antibody against the immunogen are obtained, blood iscollected from the animal and antisera is prepared. Furtherfractionation of the antisera to enrich for antibodies reactive toNeospora proteins can be done if desired. (See Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988)).

Polyclonal antisera to the BPA1 and BPA3 isolates have been produced andevaluated. The polygonal antisera are used to identify and characterizeNeospora tachyzoite and bradyzoite stages in the tissues of infectedanimals using, for instance, immunoperoxidase test procedures describedin Anderson et al. JAVMA 198:241 (1991) and Barr et al. Vet. Pathol.28:110-116(1991).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (See, Kohler and Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.

For instance, the BPA1 isolate has been used to immunize mice to obtainsensitized B cells for hybridoma production. Using these cells,monoclonal antibodies to the 48 kd and 70 kd Neospora proteins have beenobtained. The monoclonal antibodies produced are used, for instance, inELISA diagnostic tests, immunohistochemical tests, for the in vitroevaluation of parasite invasion, to select candidate antigens forvaccine development, protein isolation, and for screening genomic andcDNA libraries to select appropriate gene sequences.

Diagnosis of Neospora Infections

The present invention also provides methods for detecting the presenceor absence of Neospora in a biological sample. For instance, antibodiesspecifically reactive with Neospora can be detected using eitherproteins or the isolates described here. The proteins and isolates canalso be used to raise specific antibodies (either monoclonal orpolyclonal) to detect the antigen in a sample. In addition, the nucleicacids disclosed and claimed here can be used to detect Neospora-specificsequences using standard hybridization techniques. Each of these assaysis described below.

A. Immunoassays

For a review of immunological and immunoassay procedures in general, seeBasic and Clinical Immunology 7th Edition (D. Stites and A. Terr ed.)1991. The immunoassays of the present invention can be performed in anyof several configurations, which are reviewed extensively in EnzymeImmunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980);"Practice and Theory of Enzyme Immunoassays," P. Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B. V. Amsterdam (1985). For instance, the proteins andantibodies disclosed here are conveniently used in ELISA, immunoblotanalysis and agglutination assays. Particularly preferred assay formatsinclude the indirect fluorescent antibody assay as described in Example2.

In brief, immunoassays to measure anti-Neospora antibodies or antigenscan be either competitive or noncompetitive binding assays. Incompetitive binding assays, the sample analyte (e.g., anti-Neosporaantibodies) competes with a labeled analyte (e.g., anti-Neosporainonoclonal antibody) for specific binding sites on a capture agent(e.g., isolated Neospora protein) bound to a solid surface. Theconcentration of labeled analyte bound to the capture agent is inverselyproportional to the amount of free analyte present in the sample.

Noncompetitive assays are typically sandwich assays, in which the sampleanalyte is bound between two analyte-specific binding reagents. One ofthe binding agents is used as a capture agent and is bound to a solidsurface. The second binding agent is labeled and is used to measure ordetect the resultant complex by visual or instrument means.

A number of combinations of capture agent and labeled binding agent canbe used. For instance, an isolated Neospora protein or culture can beused as the capture agent and labeled anti-bovine antibodies specificfor the constant region of bovine antibodies can be used as the labeledbinding agent. Goat, sheep and other nonbovine antibodies specific forbovine immunoglobuliln constant regions (e.g. γ or μ) are well known inthe art. Alternatively, the anti-bovine antibodies can be the captureagent and the antigen can be labeled.

Various components of the assay, including the antigen, anti-Neosporaantibody, or anti-bovine antibody, may be bound to a solid surface. Manymethods for immobilizing biomolecules to a variety of solid surfaces areknown in the art. For instance, the solid surface may be a membrane(e.g., nitrocellulose), a microtiter dish (e.g., PVC or polystyrene) ora bead. The desired component may be covalently bound or noncovalentlyattached through nonspecific bonding.

Alternatively, the immunoassay may be carried out in liquid phase and avariety of separation methods may be employed to separate the boundlabeled component from the unbound labeled components. These methods areknown to those of skill in the art and include immunoprecipitation,column chromatography, adsorption, addition of magnetizable particlescoated with a binding agent and other similar procedures.

An immunoassay may also be carried out in liquid phase without aseparation procedure. Various homogeneous immunoassay methods are nowbeing applied to immunoassays for protein analytes. In these methods,the binding of the binding agent to the analyte causes a change in thesignal emitted by the label, so that binding may be measured withoutseparating the bound from the unbound labeled component.

Western blot (immunoblot) analysis can also be used to detect thepresence of antibodies to Neospora in the sample. This technique is areliable method for confirming the presence of antibodies against aparticular protein in the sample. The technique generally comprisesseparating proteins by gel electrophoresis on the basis of molecularweight, transferring the separated proteins to a suitable solid support,(such as a nitrocellulose filter, a nylon filter, or derivatized nylonfilter), and incubating the sample with the separated proteins. Thiscauses specific target antibodies present in the sample to bind theirrespective proteins. Target antibodies are then detected using labeledanti-bovine antibodies.

The immunoassay formats described above employ labeled assay components.The label can be in a variety of forms. The label may be coupleddirectly or indirectly to the desired component of the assay accordingto methods well known in the art. A wide variety of labels may be used.The component may be labeled by any one of several methods.Traditionally a radioactive label incorporating ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C,or ³² P was used. Non-radioactive labels include ligands which bind tolabeled antibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand. The choice of label depends on sensitivity required,ease of conjugation with the compound, stability requirements, andavailable instrumentation.

Enzymes of interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminnescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule:The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need to be labeled and the presence ofthe target antibody is detected by simple visual inspection.

B. Detection of Neospora nucleic acids

As noted above, this invention also embraces methods for detecting thepresence of Neospora DNA or RNA in biological samples. These sequencescan be used to detect all stages of the Neospora life cycle (e.g.,tachyzoites, bradyzoites, and oocysts) in biological samples from boththe bovine host and the definitive host. A variety of methods ofspecific DNA and RNA measurement using nucleic acid hybridizationtechniques are known to those of skill in the art. See Sambrook et al.,supra.

One method for determining the presence or absence of Neospora DNA in asample involves a Southern transfer. Briefly, the digested DNA is run onagarose slab gels in buffer and transferred to membranes. In a similarmanner, a Northern transfer may be used for the detection of NeosporamRNA in samples of RNA. Hybridization is carried out using labeledoligonucleotide probes which specifically hybridize to Neospora nucleicacids. Labels used for this purpose are generally as described forimmunoassays. Visualization of the hybridized portions allows thequalitative determination of the presence or absence of Neospora genes.

A variety of other nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in "Nucleic Acid Hybridization, A PracticalApproach," Ed. Hades, B. D. and Higgins, S. J., IRL Press, 1985; Galland Pardue (1969), Proc. Natl. Acad. Sci., U.S.A., 63:378-383; and John,Burnsteil and Jones (1969) Nature, 223:582-587.

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a"capture" nucleic acid covalently immobilized to a solid support andlabeled a "signal" nucleic acid in solution. The biological sample willprovide the target nucleic acid. The "capture" nucleic acid and "signal"nucleic acid probes hybridize with the target nucleic acid to form a"sandwich" hybridization complex. To be effective, the signal nucleicacid cannot hybridize with the capture nucleic acid.

The sensitivity of hybridization assays may be enhanced through use of anucleic acid amplification system which multiplies the target nucleicacid being detected. Examples of such systems include the polymerasechain reaction (PCR) system and the ligase chain reaction (LCR) system.Other methods recently described in the art are the nucleic acidsequence based amplification (NASBA™, Cangene, Mississauga, Ontario) andQ Beta Replicase systems.

An alternative means for detecting Neospora nucleic acids is in situhybridization. In situ hybridization assays are well known and aregenerally described in Angerer, et al., Methods Enzymol., 152:649-660(1987). In situ hybridization assays use cells or tissue fixed to asolid support, typically a glass slide. If DNA is to be probed, thecells are denatured with heat or alkali. The cells are then contactedwith a hybridization solution at a moderate temperature to permitannealing of labeled Neospora specific probes. The probes are preferablylabeled with radioisotopes or fluorescent reporters.

Exemplary nucleic acid sequences for use in the assays described aboveinclude sequences from the nss-rRNA sequences disclosed here. Forinstance, the primer and probe sequences disclosed in Example 4 can beused to amplify and identify nucleic acids of bovine Neospora in blood,cerebrospinal fluid and fetal fluids, as well as in frozen orformalin-fixed tissue. These primers are particularly useful for thediagnosis of neosporosis and identification of the source of Neosporaparasite stages (tachyzoites, bradyzoites and oocysts) in various animalhosts.

Pharmaceutical Compositions Comprising Neospora

A pharmaceutical composition prepared using anti-Neospora monoclonalantibodies or fragments thereof as well as Neospora proteins or theirimmunogenic equivalents can be used in a variety of pharmaceuticalpreparations for the treatment and/or prevention of Neospora infections.

The pharmaceutical compositions are typically used to vaccinate cattle,sheep, goats and other animals infected by Neospora. The compositions ofthe invention can also be used to treat the definitive host to preventthe shedding of oocysts and subsequent transfer to cattle. Thecompositions for administration to either cattle or the definitive hostcan comprise tachyzoite and/or bradyzoite antigens.

An attenuated Neospora vaccine can only be used in the absence of a riskof human infection should the milk or tissues of immunized animals beconsumed. Thus, preferred vaccines are subunit vaccines that elicitantibody and cell-mediated immunity (CMI) to antigens of bovineNeospora. Experimental evidence indicates that CMI is an importantcomponent of the protective immune response in cattle. A variety ofmethods for evaluating the specificity of the helper and cytotoxic Tcell response to selected antigens in vitro can be used. In addition, asdemonstrated below, cows infected using culture-derived tachyzoitesmount a protective immune response and prevent transplacental infectionof the fetus.

The vaccines of the invention are typically administered orally orparenterally, usually intramuscularly or subcutaneously. For parenteraladministration, the antigen may be combined with a suitable carrier. Forexample, it may be administered in water, saline or buffered vehicleswith or without various adjuvants or immunomodulating agents such asaluminum hydroxide, aluminum phosphate, aluminum potassium sulfate(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oilemulsions, oil-in-water emulsions, muramyl dipeptide, bacterialendotoxin, lipid, Bordetella pertussis, and the like. Such adjuvants areavailable commercially from various sources, for example, Merck Adjuvant6 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants areMPL+TDM Emulsion (RIBBI Immunochem Research Inc. U.S.A.). Otherimmuno-stimulants include interleukin 1, interleukin 2 andinterferon-gamma. These proteins can be provided with the vaccine ortheir corresponding genetic sequence provided as a functional operonwith a recombinant vaccine system such as vaccinia virus. The proportionof antigen and adjuvant can be varied over a broad range so long as bothare present in effective amounts.

Vaccine compositions of the invention are administered to a cattle,sheep, or goats susceptible to or otherwise at risk of infection toelicit an immune response against the antigen and thus enhance theanimals own immune response capabilities. Such an amount is defined tobe an "immunogenically effective amount." In this use, the preciseamounts depend on the judgement of the prescribing veterinarian andwould include consideration of the animals state of health and weight,the mode of administration, the nature of the formulation, and the like.Generally, on a per-dose basis, the concentration of the Neosporaantigen can range from about 1 μg to about 100 μg per bovine host. Foradministration to cattle, a preferable range is from about 100 μg toabout 1 mg per unit dose. A suitable dose size is about 1-10 ml,preferably about 1.0 ml. Accordingly, a typical dose for subcutaneousinjection, for example, would comprise 1 to 2 ml containing 0.1 to 10 mgof antigen.

A variety of vaccination regimens may be effective in immunizing cattleand other animals. Preferably, female cattle (heifers and cows) arevaccinated just prior to or at the time of breeding so as to preventabortion and reduce the possibility of congenital infections. A secondimmunization will be given 2-4 weeks after initial immunization. Calvesand adult males may also be vaccinated, if desired. Animals that havebeen previously exposed to Neospora or have received colostralantibodies from the mother may require booster injections. The boosterinjection is preferably timed to coincide with times of maximalchallenge and/or risk of abortion. Different immunization regimes may beadopted depending on the judgement of the veterinarian.

Vaccines of the invention may comprise a crude extract of Neosporatachyzoites, bradyzoites or other stages. Chemically fixed parasites orcells can also be used. As noted above, preferred vaccines comprisepartially or completely purified Neospora protein preparations. Theantigen produced by recombinant DNA technology is preferred because itis more economical than the other sources and is more readily purifiedin large quantities.

In addition to use in recombinant expression systems, the isolatedNeospora gene sequences can also be used to transform viruses thattransfect host cells in animals. Live attenuated viruses, such asvaccinia or adenovirus, are convenient alternatives for vaccines becausethey are inexpensive to produce and are easily transported andadministered.

Suitable viruses for use in the present invention include, but are notlimited to, pox viruses, such as capripox and cowpox viruses, andvaccinia viruses, alpha viruses, adenoviruses, and other animal viruses.The recombinant viruses can be produced by methods well known in theart, for example, using homologous recombination or ligating twoplasmids. A recombinant canarypox or cowpox virus can be made, forexample, by inserting the DNA's encoding the Neospora protein orfragments thereof into plasmids so that they are flanked by viralsequences on both sides. The DNA's encoding Neospora polypeptides arethen inserted into the virus genome through homologous recombination.

Preferentially, a viral vaccine using recombinant vaccinia virus isused. A vaccine prepared utilizing the gene encoding the Neosporaprotein incorporated into vaccinia virus would comprise stocks ofrecombinant virus where the gene encoding the Neospora protein isintegrated into the genome of the virus in a form suitable forexpression of the gene.

EXAMPLES Example 1

This example describes the isolation and in vitro cultivation ofNeospora from aborted bovine fetuses. The isolation of these 2 cultures(BPA1 and BPA2) is described in Conrad et al. Parasitol. 106:239-249(1993). Additional cultures (BPA3-6) were also isolated using the sametechnique, except that 1 ml (instead of 2 ml) of brain or spinal cordhomogenate was trypsinized and then incubated for 2-4 hours, rather thanovernight, on the cell monolayer. In addition, the bovine aorticendothelial cell line (CPAE: American Tissue Culture Collection #CCL209)was found to be the best cell monolayer for the cultivation of bovineNeospora. One of these cultures (BPA6) has been shown to inducebradyzoite cysts in mice.

MATERIALS AND METHODS

Pathological Examination and Immunohistochemistry of fetal Tissues

Aborted bovine fetuses submitted to the California Veterinary DiagnosticLaboratory System were necropsied using standard techniques. The brainsfrom fetuses suspected of having protozoal infections were removedaseptically from the cranium. One half of the brain was placed insterile saline (0-85% w/v) containing 1000 U/ml penicillin G and 100μg/ml streptomycin (antibiotic saline) and stored at 4° C. until adiagnosis of protozoal infection was confirmed, at which time the braincould be processed for in vitro cultivation. Multiple tissues, includingportions of the brain, liver, kidney, heart, lung, spleen,gastrointestinal tract, skeletal muscle, adrenal gland, trachea andthymus, were collected from each fetus and fixed in 10% neutral bufferedformalin for 24 h. Fixed tissues were trimmed, embedded in paraffin,sectioned, stained with hematoxylin and eosin, and examined by lightmicroscopy for the presence of lesions and parasites, as previouslydescribed (Barr et al. 1990 Vet. Pathol. 27:354-61).

Fetuses with multifocal microgliosis and/or necrosis in the brain,suggesting protozoal infection, were further examined byimmunohistochemistry for the presence of parasites in brain tissuesections using an avidin-biotin peroxidase complex procedure (VectorLaboratories, Burlingame, Calif., USA) with anti-rabbit serum to detecttissue-binding of rabbit polyclonal anti-N. caninum serum. Theimmunoperoxidase method employed was basically as described previously(Barr et al. 1991 J. Vet. Diag. Invest. 3:39-46) except that tissuesections were processed using a microprobe system (FisherBiotech,Pittsburgh, Pa., USA) and Probe-On glass slides (FisherBiotech).Aminoethylcarbazole (A.E.C. Substrate System, Dako, Santa Barbara,Calif., USA) was the chromogen.

Parasites in the tissue sections of brains from the 66th and 93rd fetus(hereafter referred to as fetus 66 and 93) were further characterized bythe same immunohistochemical procedure to test their reactivity withantisera to additional apicomplexan protozoal parasites. Tissue sectionswere incubated at room temperature for 1 h with optimal dilutions of thefollowing antisera: 1:1000 dilution of antiserum to N. caninumtachyzoites (Lindsay & Dubey, 1989 Am. J. Vet. Res. 50:1981-3), 1:50dilution of antiserum to Hammondia hammondi tissue cysts and 4 differentantisera to T. gondii (Tg1-4). Antiserum Tg1 was produced by theinfection of a rabbit with live sporulated oocysts of the ME-49 strain(Lindsay & Dubey, supra) of T. gondii and used at a 1:400 dilution.Toxoplasma gondii antiserum Tg2 (Dr J. C. Boothroyd, StanfordUniversity) was produced by immunization of a rabbit with a tachyzoitelysate of the RH strain of T. gondii and was used at a dilution of1:300. Antisera Tg3 (BioGenex Laboratories, Dublin, Calif., USA) and Tg4(I.C.N. Immunobiologicals, Lisle, Ill., USA) were developed byimmunizing rabbits with tachyzoites of the RH and H44 strains,respectively. Antiserum Tg3 was applied as supplied by the manufacturerand Tg4 was used at a 1:80 dilution. The optimal dilution chosen foreach antiserum produced a strongly positive reaction against therespective positive control parasite with no appreciable non-specific,background staining. Control tissues consisted of paraffin-embeddedsections of murine brain with N. caninum tachyzoites, murine brain withT. gondii cysts, murine spleen with T. gondii tachyzoites, murineskeletal muscle with H. hammondi cysts and bovine tongue withSarcocystis cruzi cysts (Barr et al. 1991 Vet. Path. 28:110-116).

Parasite Cultures

Stationary monolayer cultures of bovine cardiopulmonary aorticendothelial cells (CPAE: ATCC #CCL209) and M617 bovine macrophages(Speer et al. 1985 Infect. and Immun. 50:566-71) were maintained inDulbecco's Minimum Essential Medium (DMEM:GIBCO Laboratories, GrandIsland, N.Y., USA) supplemented with 10% (v/v) heat-inactivated fetalbovine serum (FBS) or heat-inactivated adult equine serum (HS), 2 mML-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (DMEM-FBS orDMEM-HS). Bovine fetal trophoblast cells (87-3) were grown as previouslydescribed by Munson et al. 1988 J. Tissue Cult. Methods 11:123-8) inDMEM supplemented with 10% (v/v) FBS, 5 μg/ml transferrin, 5 μg/mlinsulin, 5 ng/ml selenium, 10 ng/ml epidermal growth factor, 100 μg/mLstreptomycin, 100 U/mi penicillin and 0.25 μg/mi amphotericin B(DMEM-FBS*). Control cultures of T. gondii (RH strain provided by Dr J.Boothroyd) and N. caninum (NCl; Dubey et al. 1988 J Am Vet Med Assoc193:1259-1263) were maintained in the CPAE and M617 monolayer cultures.Parasite-infected and uninfected monolayer cultures were maintained in25 or 75 cm² flasks incubated at 37° C. with 5% CO₂. The culture mediumwas changed 3 times weekly. Established parasite cultures were passagedto uninfected monolayers when 70-90% of cells were infected. To passageparasites, the infected monolayer was removed from the flask by scrapinginto the media and passed 3 times through a 25-gauge needle to disruptthe cells. The cell suspension was then diluted from 1:2 to 1:8 in freshcomplete media and added to a confluent, uninfected monolayer.

After protozoal infection was confirmed by histology, fetal brain tissuethat was stored at 4° C. for a variable period of time in antibioticsaline was processed for in vitro cultivation. In all cases whereisolates were obtained in vitro, tissue cysts were seen in tissuesections of bovine fetal brain. Half of the fetal brain in approximately25 ml of antibiotic saline was ground with a pestle and mortar andfiltered through sterile gauze. Aliquots of 2 ml of brain homogenatewere placed in 10 ml of 0.05% (v/v) trypsin and incubated at 37° C. for1 h. After trypsin digestion, the material was pelleted bycentrifugation at 600 g for 10 min. The supernatant was discarded andthe pellet was resuspended in 1-3 ml of either DMEM-HS or DMEM-FBS.Brain from fetus 66 was prepared for culture 48 h after submission and 1ml of trypsinized brain suspension was dispensed into a 25 cm² flask ofbovine 87-3 trophoblast cells. Brain from fetus 93 was processed 10 daysafter submission when half of the trypsinized brain was placed in a 25cm² flask of 87-3 trophoblast cells and the remainder in a 75 cm² flaskof endothelial cells. After incubation overnight, the brain suspensionfrom both fetuses was removed from the flask and the monolayers werewashed 3 times with the appropriate media before adding 5-10 ml of freshmedia. Cultures were maintained as described above and examined with aninverted microscope for the presence of parasites.

Immunohistochemistry of Tachyzoites in vitro

Antigenic reactivity of the two in vitro isolates from aborted bovinefetuses was compared to that of tachyzoites from control cultures of T.gondii and N. caninum. Tachyzoites of each isolate were harvested duringlogarithmic growth by scraping the infected CPAE monolayer from a 25 cm²tissue culture flask. Monolayer cells were disrupted by repeated passagethrough a 25-gauge needle. The suspension was passed through a 5 μmfilter to remove cellular debris and pelleted by centrifugation at 1500g for 10 min. After removing the supernatant fraction, the pelletedtachyzoites of each isolate were resuspended in DMEM-HS and inoculatedinto each of the wells on two 4-chambered tissue culture slides(Lab-Tek, Nunc, Naperville, Ill., USA). Each of the 4 chambers on theseslides were seeded 24-48 h prior to parasite inoculation with CPAE cellsso that the monolayers were 60-80% confluent at the time of infection.The appropriate slides were inoculated with the slower growing bovinefetal isolates first to allow the parasites to grow for 48 h, whereasthe isolates of T. gondii and N. caninum were cultivated on slides for24 h before being processed for immunohistochemical evaluation.

To prepare the parasite cultures for immunohistochemistry, culturesupernatants were removed with monolayers remaining adherent to glassslides. These slides were fixed in 100% methanol (4° C.) for 10 min andallowed to air dry completely before being washed 3 times for 5 min eachin physiologically buffered saline (PBS:pH 7.2), incubated for 10 min in3% (v/v) hydrogen peroxide in methanol, washed again 3 times for 5 mineach in PBS and incubated for 30 min with 20% goat serum to blocknon-specific antibody binding sites. Each slide was then incubated for 1h with 3 wells containing different primary antiserum and 1 well servingas a negative control with a pre-infection rabbit serum. The optimalantisera dilutions were selected to produce a strongly positive reactionagainst the homologous culture-derived antigen with no appreciablenon-specific, background staining. The dilutions of antisera used forstaining parasites in vitro were 1:3000 for N. caninum, 1:800 for Tg1,1:40 for Tg2, 1:1 for Tg3, 1:2000 for Tg4 and 1:50 for H. hammondi.Slides were washed 3 times for 5 min each in PBS and the secondaryantibody and conjugate were applied as described above for the tissuesections except that the slides were processed manually and thechromogen was applied for only 2 min.

RESULTS

The first parasite isolate (BPA1) was obtained from fetus 66 which wasestimated to be approximately 4 months gestational age and in relativelygood postmortem condition at the time of necropsy. Significant grosslesions were restricted to focal epicardial petechiae. On histologicalexamination there were infrequent, random, small foci of gliosis and 5protozoal cysts were seen in sections of the fetal brain. The tissuecysts ranged from 8 to 10 μm in diameter and had distinct, thin walls(<1 μm) surrounding at least 25-40 closely packed bradyzoites. Inaddition there were scattered mononuclear inflammatory cell infiltratesin the heart, portal tracts of the liver and throughout the renalcortex. In the lung, macrophages and neutrophils were present withinalveolar septa, adjacent to bronchioles and free in the lumen ofbronchioles and alveoli. Escherichia coli and Proteus spp. were isolatedfrom the lung, liver, spleen and abomasal contents of this fetus.

The second isolate (BPA2) was obtained from fetus 93 which had anestimated gestational age of 6 months and was mildly autolyzed.Histological examination revealed infrequent random foci of gliosis inthe brain with adjacent capillaries that had prominent hypertrophiedendothelium. There was also a mild diffuse meningeal infiltrate ofmononuclear cells, consisting predominantly of lymphocytes withoccasional plasma cells. Four randomly located protozoal tissue cystswere found in the brain; one being located adjacent to a focus ofgliosis. The cysts were 8-13 μm in diameter with at least 25-50bradyzoites. Two of the cysts had thick (1-2 μm) walls. Focal, mixedmononuclear inflammatory cell infiltrates were also present in skeletalmuscle and in the renal cortex.

Table 1 summarizes the immunoreactivity of protozoal tissue cysts in thebrains of fetus 66 and fetus 93 with different polyclonal antisera andcompares these antigenic reactions to those of N. caninum, T. gondii, H.hammondi and S. cruzi. The protozoal cysts in fetuses 66 and 93 reactedmost strongly to N. caninum antiserum and had weaker reactions to H.hammondi antiserum. With both antisera, staining was predominantly tothe cyst wall with some staining of zoites within the cysts. Overall,the reactivity of cysts in the two bovine fetuses was more similar tothe reactivity of N. caninum tachyzoites than to that of T. gondiitachyzoites or cysts, H. hammondia cysts or S. cruzi cysts.

                                      TABLE 1    __________________________________________________________________________    Reactivity of tissue cysts and tachyzoites with rabbit polyclonal    antisera    against different parasites in an immunoperoxidase test                      Antisera                      Neospora                           Hammondia                                 Toxoplasma gondii    Parasite antigen               Tissue caninum                           hammondi                                 Tg1                                    Tg2                                       Tg3                                          Tg4    __________________________________________________________________________    Fetus 66 cysts               Bovine brain                      ++*  ++*   ±                                    -  -  -    Fetus 93 cysts               Bovine brain                      ++*  +*    -  -  -  -    Neospora caninum               Mouse brain                      +++  -     ++ -  -  -    tachyzoites    Toxoplasma gondii               Mouse spleen                      -    ++    +++                                    +++                                       ±                                          ±    tachyzoites    Toxoplasma gondii               Mouse brain                      +    +++   +++                                    +  ++ ++    cysts    Hammondia hammondi               Mouse muscle                      ± +++   +++                                    -  +  ++    cysts    Sarcocystis cruzi               Bovine -    -     -  -  -  -    cysts      muscle    __________________________________________________________________________     *Primarily cyst wall staining.

In approximately 14 months, over 100 fetuses were submitted specificallyas protozoal abortion suspects from dairies in California. Parasiteisolations were attempted with brains from the 49 fetuses which hadNeospora-like protozoa identified by immunohistochemistry. The firstevidence of parasite growth in these cultures was detected in the 87-3cell line 34 days after inoculation of brain tissue from fetus 66(isolate BPA1). The next successful in vitro isolation was apparent whentachyzoites were first observed in 87-3 and CPAE cultures on day 15after inoculation with brain tissue from fetus 93 (isolate BPA2). Incultures of both isolates, parasites first appeared as small clusters ofintracellular tachyzoites in pairs or random groups. Extracellulartachyzoites were seen escaping from the bovine cell monolayers andmoving by gliding and twisting in the culture medium. On Giemsa-stainedsmears of infected monolayers, extracellular tachyzoites were 1.5-2.5 μmwide at the nucleus and 6-8 μm long. The number of tachyzoite clustersand number of tachyzoites in each cluster increased gradually in thecultures as they became established with continuous parasite growth.Generally, parasite clusters contained approximately 10-100 tachyzoites.Growth of both isolates was maintained in cultures of 87-3, CPAE, orM617. However, the best growth was observed in the 87-3 and CPAEcultures. Within 2-3 months of establishment, the cultures were passagedweekly whenever approximately 80-95% of the bovine monolayer cells wereinfected with tachyzoites. Routinely, the established BPA1 and BPA2cultures were passaged by adding a 1:8 dilution of needle-passagedmonolayer in fresh media to cultures of uninfected bovine monolayers. Bycomparison, in our laboratory, cultures of T. gondii (RH isolate) areroutinely passaged at a 1:200 dilution and N. caninum (NC-1 isolate)cultures are passaged at a 1:10 dilution every 2-3 days. As of mid-May1992, cultures of the BPA1 and BPA2 isolates had been maintained withcontinuous growth for 10 and 6 months, respectively.

The results of an antigenic comparison of in vitro cultivatedtachyzoites of BPA1 and BPA2 to those of cultivated N. caninum and T.gondii tachyzoites are shown in Table 2. The reactions of the bovinefetal isolates to the different antisera were similar to thatdemonstrated by N. caninum, and distinctly different from the pattern ofreactivity observed with T. gondii tachyzoites (Table 2).

                  TABLE 2    ______________________________________    Reactivity of in vitro cultivated tachyzoites with rabbit polyclonal    antisera    against different parasites in an immunoperoxidase test            Antisera                       Toxoplasma gondii    Parasite  Neospora Hammondia    antigen   caninum  hammondi  Tg1  Tg2  Tg3  Tg4    ______________________________________    Fetus 66 (BPA1)              +++      -         -    +    -    -    Fetus 93 (BPA2)              +++      -         ± +    -    -    Neospora  +++      -         -    +    -    -    caninum    Toxoplasma              -        +++       +++  +++  +++  +++    gondii    ______________________________________

By transmission electron microscopy, tile in vitro tachyzoites ofisolates BPA1 and BPA2 were morphologically similar. Therefore, thefollowing ultrastructural description applies to both isolates.Individual tachyzoites or clusters of multiple tachyzoites were usuallylocated within a parasitophorous vacuole in the cytoplasm of bovinemonolayer cells. Tachyzoites had a pellicle consisting of a complex of 2inner membranes beneath a plasmalemmal membrane, a prominent nucleus inthe central or posterior portion of the tachyzoite, 1 to 3 profiles oflong tubular cristate mitochondria, a Golgi complex, rough and smoothendoplasmic reticulum, single- or multiple-membraned vesicles, andnumerous free ribosomes. Ultrastructural apical features characteristicof apicomplexan parasites were present in tachyzoites of both isolates,including a polar ring which gave rise to 22 longitudinal subpellicularmicrotubules, a cylindrical or cone-shaped conoid within the polar ringand numerous electron dense rhoptries. The number of rhoptries visiblein individual tachyzoites varied greatly and was dependent to someextent on the plane of section. A maximum of 24 rhoptries was counted inthe anterior end of transversely sectioned tachyzoites. Rhoptries werenot seen posterior to tachyzoite nuclei. In longitudinal sections,rhoptries were elongated, club-shaped structures with narrow, densenecks that extended into the conoid. As many as 14-32 electron-densebodies were observed primarily posterior to the nucleus. Smaller numbersof these dense bodies were found anterior to the nucleus. Unlikerhoptries, dense bodies were generally round or oval in longitudinalsections. Large numbers of micronemes were seen in the anterior end oftachyzoites and only rarely observed posterior to the nucleus. Themicronemes were most often arranged in organized arrays or sheets thatwere orientated parallel to the pellicular membrane or longitudinal axisof the tachyzoite. The number varied greatly in individual tachyzoitesbut as many as 60-100 micronemes were counted in longitudinal or obliquesections of selected tachyzoites. In addition, a single micropore,located anterior to the nucleus was seen in some tachyzoites. Parasitesmultiplied by endodyogeny and many were observed in the process offorming 2 progeny zoites within a single tachyzoite. Rarely, as many as4 zoites with intact nuclei were seen in division but still attached toeach other at the posterior end.

A concerted effort focused on the in vitro isolation of Neospora-likeprotozoal parasites from cattle after studies showed that they were themajor diagnosed cause of abortion in California dairy cattle.Histologically, the two bovine fetuses from which isolates were firstobtained in 1991 had compatible lesions, including multifocalnon-supportive encephalitis, and protozoal tissue cysts like those seenin other natural infections of Neospora-like protozoa in bovine fetuses.The immunological reactivity of tissue cysts in the brains of fetus 66and fetus 93 was also similar to that seen with many of the cysts innaturally infected fetuses which had strong reactions with N. caninumand H. hammondi antisera and occasional reactions to some of the T.gondii antisera.

Isolation of these Neospora-like protozoal parasites from aborted bovinefetuses was difficult because fetuses are generally moderately toseverely autolyzed at the time of abortion and protozoal tissue cystsare present in a relatively small proportion of the infected fetuses.Previous ultrastructural studies suggested that most of the protozoalcysts in these fetal tissues were affected by autolysis and wereprobably non-viable (Barr et al. 1991 Vet. Path. 28:110-16). The twofetuses from which isolates were obtained were in comparatively goodpostmortem condition. This fact, plus the relatively large number ofcysts in these fetuses may have been critical factors in the successfulisolation of the protozoal parasites. In addition, the isolation methodswere modified. In particular, a longer period of trypsinization inpreparing the brain material for cultivation, the overnight incubationof the brain homogenate on the monolayer, and use of the 87-3 bovinetrophoblastic cell line for the initial parasite isolation weremodifications in these procedures which appeared to be particularlyhelpful in obtaining the BPA1 and BPA2 isolates. Parasite growth wasbest maintained in 87-3 and CPAE monolayer cells. This contrasts with N.caninum and Hammondia heydorni which are reported to grow better inbovine monocyte cells (Speer et al. 1988 Infect. and Inmmun. 50:566-71).

In comparing these bovine protozoal isolates to those of T. gondii andN. caninum that have been re-isolated from mouse brains, it was foundthat the bovine protozoal isolates grew more slowly during isolation andafter establishment of continuous growth. Whether this reflects adifference in the virulence of the organisms or a difference inadaptation to culture remains to be determined.

By light microscopy, tachyzoites of the bovine isolates weremorphologically similar to in vitro tachyzoites of T. gondii and N.caninum. Cultivated tachyzoites of the bovine isolates had similarimmunohistochemical reactions to tachyzoites of N. caninum, reactingstrongly with N. caninum anti-serum and weakly to serum Tg1 which wasproduced by immunization of a rabbit with tachyzoite lysates of T.gondii. These antigenic reactions were distinctly different from thoseseen with culture-derived tachyzoites of T. gondii. Differences inantigenic reactivity of the cultivated BPA1 and BPA2 tachyzoites, ascompared to those of tissue cysts in the source fetuses, could beexplained by the stage-specific antigen expression of the differentparasites and variations in the methods used to produce the antisera(i.e. immunization with cysts, oocysts or tachyzoite lysates). Forexample, tissue cyst wall antigens that reacted with antiserum to H.hammondi appeared to be lacking on tachyzoites of the bovine isolates invitro. Unfortunately, a direct comparison of different parasite stageswas not always possible since tachyzoites were not identified in thebrains of the two bovine fetuses and true cysts have not been observedin the BPA1 or BPA2 cultures. Similarly, N. caninum tissue cysts andculture-derived H. hammondi tachyzoites were not available forcomparison. Differences in antigenic expression may also be affected byhost-specific factors. To evaluate this possibility, efforts are underway to obtain material from cattle, dogs, rats, cats and mice that havebeen experimentally infected with N. caninum or the bovine isolates soas to make a direct comparison of antigenic reactivity of the parasitesin the same host species.

Thus far, characterization of the in vitro isolates from the two abortedbovine fetuses has shown that these parasites are antigenically and/orultrastructurally distinct from T. gondii, H. hammondi, S. cruzi,Besnoitia spp. and Frenkelia spp. These isolates most closely resembleN. caninum parasites which have been most extensively studied in the USAand Scandinavia. The similarity between these parasites indicates thatthe BPA1 and BPA2 isolates belong to the genus Neospora. At presentlittle is known about the life-cycle, including the definitive host, ofthese Neospora parasites in dogs or cattle. A better understanding ofthe biology of these parasites is essential to determine their taxonomicrelationship to each other and to other apicomplexan parasites.

Example 2

This example describes an indirect fluorescent antibody (IFA) test forthe detection of parasite-specific antibody responses in cattle thatwere naturally or experimentally infected with Neospora parasites. Themethods used here are generally as described in Conrad et al. 1993 J.Vet. Diagn. Invest. 5:572-578 (1993).

MATERIALS AND METHODS

Parasites and Antigen Slide Preparation

Antigen slides were prepared using tachyzoites of the BPA1 describedabove. Culture media consisted of Dulbecco's Minimum Essential Medium(DMEM) supplemented with 10% (v/v) heat-inactivated adult equine serum,2 mM L-glutamine, 50 U/ml penicillin and 50 ug/ml streptomycin(DMEM-HS). Tachyzoites of Toxoplasma gondii (RH isolate; kindly providedby Dr. J. Boothroyd) were obtained from CPAE monolayer cultures grown inthe same medium except that 10% (v/v) heat-inactivated fetal bovineserum was used instead of equine serum. Parasite-infected cultures weremaintained in 25 or 75 cm² flasks incubated at 37° C. in an atmosphereof 5% CO₂.

Parasites were harvested for antigen preparation when ≧80% of the CPAEcells in the culture flask were infected with clusters of tachyzoites.The infected monolayer was removed from the flask by scraping into themedium and then passed 3× through a 25 gauge needle to disrupt thecells. The suspension was passed through a 5 μm filter to removecellular debris and tachyzoites were pelleted by centrifugation at1300×g for 10 min. After removing the supernatant, the pellet was washedtwice in sterile phosphate buffered saline pH 7.2 (PBS) and thenresuspended in a modified PBS saline (137 mM NaCl, 3 mM KCl, 3 mM Na₃ C₆H₅ O₇.2H₂ O, 0.4 mM NaH₂ PO₄.H₂ O, 12 mM NaHCO₃, 6 mM glucose) to afinal concentration of approximately 2,000/μl. Aliquots of 10 μl oftachyzoite suspension were dispensed into each 4 mm well on 12-wellheavy teflon coated (HTC) antigen slides. Slides were air-dried at roomtemperature and stored at -70° C.

Cattle

Test sera was obtained from naturally infected cows that abortedNeospora-infected fetuses, as well as congenitally infected calves. Inaddition, sera was obtained from two pregnant heifers that wereexperimentally infected at approximately 120 days gestation withtachyzoites of the BPA1 isolate derived from CPAE cultures. Tachyzoiteswere obtained from cultures using the procedure described for harvestingtachyzoites for antigen preparation except that the parasites were notwashed in PBS and only the inoculum given to each heifer intravenouslywas filtered to remove cellular debris. After centrifugation,tachyzoites were resuspended in DMEM and administered by inoculation toeach heifer so that 3×10⁶ tachyzoites were given IV and 5×10⁶ were givenIM. A control heifer from the same herd and at the same stage ofgestation was inoculated with an equivalent amount of uninfected CPAEcell culture material which was prepared and administered using the sameprocedures as for the infected heifers. Natural or experimentalinfections were confirmed by identification of Neospora tachyzoitesand/or tissue cysts in fetal or calf tissues using an immunoperoxidasetest procedure (Anderson et al. (1991) J Am Vet Med Assoc 198:241-244and Barr et al. (1991) Vet Path 28:110-116).

For serological comparison with samples from Neospora-infected cattle,sera were obtained from the following additional sources: 1) cows thataborted fetuses which did not have lesions or parasites typical ofNeospora infections, 2) weak calves that were suspected of havingNeospora infections, but showed no lesions or parasites on post-mortemhistopathologic examination, 3) 20 heifers that were purchased asweanlings from a closed beef herd in Todd County, Nebr. and maintainedunder strict isolation, on range conditions at the Agricultural ResearchDevelopment Center, University of Nebraska-Lincoln in Mead, Nebr., 4) 20pregnant heifers that were maintained on pasture in California and 5) 21adult beef bulls or cows that were originally on pasture and thenmaintained in the same feedlot as the experimentally infected heifers.

Serum Collection and Testing

Test and control sera were obtained from blood samples that werecollected by venipuncture into vacutainer tubes without anticoagulant.After storage at 4° C. for 2-12 hr, the blood was centrifuged at 500×gfor 10 min and the serum was removed. Sera was stored either at 4° C.for <48 hr or frozen at -70° C. until tested.

Antigen slides were thawed at room temperature immediately prior to use.Sera were initially titrated in 2-fold dilutions from 1:40 to 1:40,960to determine the end-point titer. Ten ul of diluted test or control serawere placed in separate wells on the antigen slides. Slides wereincubated at 37° C. for 1 hr in a moist chamber, washed 3× for 5 mineach in PBS, and then tapped gently to remove excess PBS.Fluorescein-labeled affinity-purified rabbit anti-bovine IgG diluted1:500 in PBS was added in 10 ul aliquots to each well. Slides wereincubated at 37° C. for 30 min, washed 3 times with PBS for 5 min eachwash, tapped to remove excess PBS, cover-slipped with buffered glycerol(25% w/v! glycerine in TRIS-HCL:pH 9.0), and examined at 200magnification using a fluorescence microscope. The end-point titer wasthe last serum dilution showing distinct, whole parasite fluorescence.

RESULTS

Natural Infections

Sera collected at the time of abortion from 64 cows were tested forserologic reactivity to Neospora antigen (isolate BPA-1) using the IFAtest procedure. Aborted fetuses from 55 of these cows had nonsuppurativeencephalitis and/or myocarditis which was consistent with a protozoalinfection. In addition tachyzoite and/or cyst stages of Neospora wereidentified by immunohistochemistry in the tissues of these 55 fetuses(Table 3). In the remaining 9 fetuses there was no indication ofencephalitis and/or myocarditis and no detectable protozoal parasites.All of the cows that aborted Neospora-infected fetuses had titers of 320to 5,120 to Neospora antigens (Table 3). Eight of the cows that abortedfetuses with no detectable Neospora parasites had titers ≦160 and onehad a titer of 320.

                  TABLE 3    ______________________________________    Titers of cow sera collected after abortion of Neospora-infected    fetuses to bovine Neospora (BPA 1 isolate) antigens.    Neospora  Number  Neospora tissue stages in fetus    titer     of cows cysts    tachys                                     cysts & tachys    ______________________________________     320       1      0        1     0     640      12      2        8     2    1280      12      2        10    0    2560      15      2        11    2    5120      15      5        7     3    ______________________________________

Six of the cows that aborted Neospora-infected fetuses were maintainedon their 4 dairies of origin so that these cows could be testedrepeatedly over a 6 to 12 month period to determine changes in theNeospora titer. Peak titers of 640 to 2,560 were apparent within thefirst 20 days after abortion in all of the cows (FIGS. 1 & 2).Subsequently, the titers of 4 of the cows (FIG. 1, cows 9, 970 & 522;FIG. 2, cow 578) decreased to 640, whereas the titers of cow 3 (FIG. 1)and cow 1328 (FIG. 2) dropped to 160 within 150 days post-abortion. Cows578 and 1328 were rebred and became pregnant again within approximately50 to 70 days of aborting Neospora-infected fetuses. When these cowswere approximately 4 to 5 months pregnant their Neospora titersincreased to their original peak levels of 1,280 and remained at thislevel until the cows gave birth to full-term calves (FIG. 2). The calfborn to cow 1328 had a pre-colostral titer of 20,480 and twin calvesborn to cow 578 both had precolostral titers of 10,240 to the bovineNeospora isolate. Upon necropsy at 2 to 6 days of age, these calvesshowed mild nonsuppurative encephalomyelitis or focal mononuclear cellinfiltrates the brain parenchyma. Neospora tissue cysts were seen inassociation with inflammatory lesions in all 3 calves. Thepost-colostral titers of sera taken from each calf prior to euthanasiawere the same as their precolostral titers.

Serologic titers were determined for four additional calves that werediagnosed as having congenital Neospora infections based on the presenceof characteristic cyst stages in the brain and/or spinal cord whichreacted immunohistochemnically with antisera to the BPA-1 bovineNeospora isolate. Neospora was isolated from the brains and/or spinalcords of calves 1-3 and the parasites were grown continuously in vitro,using a previously described method for isolation of Neospora fromaborted bovine fetuses. At necropsy, calves 1 and 2 had Neospora titersof 20,480, calf 3 had a titer of 10,240 and calf 4 had a titer of 5,120.Sera collected-from the dam of calf 4 at calving had a Neospora titer of2,560. Precolostral calf sera and sera from the dams of calves 1-3 werenot available for testing.

The titers observed in the 7 congenitally infected calves with confirmedNeospora infections were markedly greater than those obtained with serafrom 4 weak 1-5 day old calves which were suspected of having Neosporainfections, but showed no histopathologic evidence of characteristiclesions or parasites on post-mortem examination. One of these uninfectedcalves had a titer of 160, while the others had titers <80 to bovineNeospora antigens. Whether or not these calves had received colostrumwas not known.

Experimental Infections

Repeated sera samples taken from the 3 pregnant heifers prior toexperimental inoculation on day 43 had titers of <80 to Neospora BPA1antigens. The 2 heifers that were infected with culture-derivedtachyzoites of the BPA1 bovine isolate developed Neospora titers of 640by day 9 and 1,280 by day 18 after parasite inoculation (FIG. 3). Theheifer that received uninfected cell culture material had titers <80 toNeospora antigens throughout the experiment. She was euthanitized 32days after inoculation to remove her fetus which was viable,histologically normal and uninfected, with no detectable titer toNeospora. Peak titers for both infected heifers were detected 32 daysafter parasite inoculation, at which time the fetus of heifer 413 wasremoved by caesarian section. Histologically, the fetus had inflammatorylesions and numerous Neospora tachyzoites present in its central nervoussystem. In addition, Neospora tachyzoites were isolated from fetaltissues and grown continuously in cell culture. Sera collected from thefetus had a titer of 640 to Neospora antigens. After her fetus wasremoved, the Neospora titer of heifer 413 fluctuated between 1,280 and5,120, until day 193 post-infection when it dropped to 640 (FIG. 3).Heifer 416 calved 158 days after parasite inoculation at which time shehad a Neospora titer of 1,280 (FIG. 3). The calf had a precolostralNeospora titer of 10,240 which was the same as the sample which wascollected 2 days later, after ingestion of the dam's colostrum.Clinically the calf appeared normal except that it had decreasedconscious proprioception in all 4 limbs when examined prior toeuthanasia at 2 days of age. There were minimal histological lesions,consisting of focal gliosis in the central nervous system, but noparasites were detected in fetal tissues.

Uninfected Cattle

Fifty three of the 61 (87%) adult cattle tested which had no history ofNeospora infection had titers ≦80, and all but one animal had titers≦160 to both Neospora and Toxoplasma antigens (Table 4). The pasturedcattle that were moved and subsequently maintained under feedlotconditions did not have higher serologic titers to tachyzoites of bovineNeospora or Toxoplasma gondii than those kept on pasture. End-pointtiter determinations of all samples from infected or uninfected cattlewere always based on whole tachyzoite fluorescence. However, in testingthe apparently uninfected animals, sera samples from 3 of the cows and 7of the bulls that were housed in the feedlot at the University ofCalifornia at Davis (UCD feedlot) had parasite fluorescence which wasrestricted to the apical end of the parasite. This reaction wasparticularly marked with the 7 sera from bulls which had apicalfluorescence titers of 160 to 320 to both Neospora and Toxoplasma, whilethe whole parasite fluorescence titer was ≦80.

                  TABLE 4    ______________________________________    Titers of sera from cattle with no evidence of Neospora infection    to bovine Neospora (BPA1 isolate) and Toxoplasma gondii.                       Neo     Number Toxo  Number    Cattle  Location   titer   positive                                      titer positive    ______________________________________    20 heifers            Nebraska   ≦80                               16     ≦80                                            14            pasture    160     3      160   6                       320     1    20 heifers            California ≦80                               17     ≦80                                            13            pasture    160     3      160   3                                      ND    4    9 cows  UCD feedlot                       <80     9      ≦80                                            7                                      160   2    12 bulls            UCD feedlot                       ≦80                               11     ≦80                                            10                       160     1      160   2    ______________________________________

FIG. 4 shows the serologic titers of the 61 uninfected adult cattle plusthe 9 cows that aborted fetuses without evidence of Neospora infectionscompared to the titers of Neospora-infected cows at the time of abortionor calving. Although a majority of the infected cattle had titers ≧1280to Neospora and most of the cattle that had no evidence of infection hadtiters ≦80, there was some overlap between these groups in the 160 to640 titer range (FIG. 4).

Example 3

This example describes isolation of DNA encoding nss-rRNA (SEQ ID NO:1).

Parasites

Bovine Neospora isolates BPA-1 BPA-2, BPA-3, BPA-4, BPA-5 were used forDNA isolation. Parasites were harvested for DNA preparation when >80% ofthe CPAE cells were infected with large clusters of tachyzoites. Theinfected monolayer was removed from the flask by scrapping. Thetachyzoites in tissue culture media were pelleted by centrifugation atroom temperature, 1300×g for 10 minutes. The supernatant was removed andthe pellet was resuspended in 10 ml sterile physiologically bufferedsaline (PBS: pH 7.4), passed through a 25 gauge needle three times todisrupt the CPAE cells, and then filtered through a 5 um disc filter(Gelman Sciences, Acrodisc) to remove cellular debris. The filteredmaterial was pelleted at 1300×g for 10 minutes and washed in PBS (pH7.4). The supernatant was removed and the tachyzoite pellet was storedat -70° C. until used. Uninfected CPAE monolayer cells were processed bythe same procedure and used as controls.

Two methods were used to prepare the DNA from the tachyzoites andcontrol CPAE cells. Initially, DNA was prepared as follows. Briefly, theparasite or control cell pellets were suspended in 1.0 ml STE with 0.5%SDS treated with proteinase K (100 μg/ml) and RNAase (100 μg/ml) thenextracted twice with phenol, once with phenol-chloroform-isoamylalcohol, and once with chloroform-isoamyl alcohol. DNA was subsequentlyprecipitated with ethanol, dried and resuspended in TE buffer. Other DNAsamples were prepared with the Isoquick DNA Extraction kit (Microprobe,Corp., Garden Grove, Calif.) following manufacturer's directions.

DNA preparations were electrophoresed in 0.8% (w/v) agarose (FMC)Bioproducts) in 0.5M Tris/borate/EDTA (TBE) buffer (89 mM Tris, 89 mMboric acid, 2 mM EDTA) gels stained with ethidium-bromide (0.5 ug/ml)and examined under ultraviolet (UV) light.

Amplification of rRNA Gene Sequences

DNA sequences were amplified by the polymerase chain reaction (PCR)using a programmable thermal cycler (Perkin-Elmer). Reactions wereperformed in 50 to 100 ul volume samples containing approximately 50-100ng of DNA template, 50 mM Tris buffer (pH 8.3), 1.0 mM MgCl₂, 200 mM ofeach of the four deoxynucleoside triphosphates, 0.5 U of Taq polymerase(Promega) and 100 pM of universal primer A (5'CCG AAT TCG TCG ACA CCTGGT TGA TCC CCG ACG ACC GTG GTC TGA ACG GGA G' (SEQ ID NO: 2)) andprimer C (5'GGG CCC TAG GTG GCG CCG ACG ACC GTG GTC TGA ACG GGA G 3'(SEQ ID NO: 3). The PCR cycling parameters consisted of a single step at94° C. for 3 minutes followed by 30 cycles denaturation at 94° C. for 1minute, 1 minute of annealing at 55, and 2 minutes of extension at 72with a final extension step of 7 minutes. The PCR amplification productwas an approximately 550-bp sequence from the 5' end of the nss-rRNAgene. A more extensive 1.8 kb sequence of the nss-rRNA gene wasamplified from BPA1 DNA using universal primer A and primer B (5' CCCGGG ATC CAA GCT TGA TCC TTC TGC AGG TTC ACC TAC 3' (SEQ ID NO: 4)).

These reactions were performed using 50 ul reaction samples thatcontained 100 pM of each primer, 1 mM MgCl₂, 50-100 ng of template DNA,50 mM Tris buffer (pH 8.3), 1.0 mM MgCl₂, a 200 mM of each of the fourdeoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP), and 0.5 U ofTaq polymerase (Promega). The amplification cycles were performed by aninitial denaturation step at 94° C. for 3 minutes followed by 45 cyclesof denaturation at 94° C. for 1 minute, 1 minute of annealing at 55° C.,and 4 minutes of extension at 72° C. with the final extension step for 7minutes. For both amplification reactions, the stationary CPAE cell DNAwas used as the negative template control. Sterile water was used forthe PCR reaction condition controls. Aliquots of each PCR product weresized by comparison with DNA standards (Bioventures, Inc. Tenn.) afterelectrophoresis through a 3% (w/v) NuSieve, 1% (w/v) SeaKem agarose gel(FMC BioProducts, Rockland Me.) stained with ethidium bromide andvisualized under UV light.

Amplification products from 3 to 5 reactions were pooled prior topurification to reduce the possibility of any nucleotidemisincorporation errors by the Taq polymerase during the elongation stepof the newly synthesized complement strain. The PCR amplificationproducts were purified either by gel electroelution or by spin-columns.Two different spin-columns were used at different times. First, MagicPCR Prep DNA purification System (Promega Corp.) was used followingmanufacturer's directions. Briefly, the products were electrophoresedthrough a low temperature melting agarose (Low Melt Agarose, FMCBioproducts,). The DNA was visualized in the gel by ethidium bromidestaining and the DNA band was excised into an eppendorf tube. Theagarose and DNA were heated (70° C.) to melt the agarose. The DNA wasseparated from the agarose using columns and reagents provided in thekit. Later simpler and less time intensive methods were used bypurifying the PCR products using the PCR Select 11 (5 Primer-3 Prime,Inc) columns which do not require electrophoresis and excision of theproduct from low melt agarose.

DNA sequencing of the purified PCR products were performed followingmanufacturer's instructions for the PCR Cycle Sequencing System (BRL dsDNA Cycle Sequencing System). The cycling parameters consisted of a twostep program after complete denaturation at 95° C. for 3 minutes. Thefirst program step amplified DNA using 20 cycles that included 30seconds for denaturation, 30 seconds for annealing, and 1 minute ofextension. The second program step alternated between denaturation (95°C.) and extension (72° C.) only. Initially, the universal primers (A, Cor B) were used to obtain the first nucleotide sequence data from whichinternal primers could be constructed and used to amplify internalsequences. All sequencing primers were 5' labeled with adenosine 5' Y³²P! triphosphate (Amersham). Reactions were heated to 95° C. for 5minutes prior to loading onto either a 6% (w/v) or 8% (w/v)polyacrylamide, 8M urea (0.4 mm thick) non-gradient gel using a Model S2Sequencing Gel Apparatus (GIBCO BRL, Gaithersburg, Md.). The sequencinggels were fixed in 10% acetic acid and 10% methanol to remove the ureaprior to transfer of the sequencing products in the gel onto filterpaper. The gels were dried using a gel drying apparatus (Biorad GelDrier, X) for 1 to 2 hours at 70°-80° C. The membrane filters wereautoradiographed using Kodak X-OMAT X-ray film.

DNA Sequence Analysis

DNA sequences were constructed from at least 3 separate reactions toensure the accuracy of the nucleotide sequence data obtained.Autoradiographs of the sequencing products were read using HibioMacDNASIS DNA and Protein Sequence Analysis System (Hitachi SoftwareEngineering Co,). This program and the GCG programs (SEQED, FragmentAssembly, Lineup, and Pretty) (University of Wisconsin Genetics ComputerGroup) on a VMS system facilitated the construction of the DNAsequences.

Example 4

The DNA prepared in Example 3 was used to design primers and probes forthe detection of Neospora. The protocol used was as follows.

Oligonucleotide PCR Primers

1) Bovine Neospora Forward Primer (5'-AAGTATAAGCTTTTATACGGCT-3' (SEQ IDNO: 5)

2) Bovine Neospora Reverse Primer (5'-CACTGCCACGGTAGTCCAATAC-3' (SEQ IDNO: 6)

DNA amplification was carried out in a total volume of 50 μl. Thereaction mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM potassiumchloride, 0.1% Triton X-100, 1.0 mM magnesium chloride, 200 mM of eachdeoxynucleoside triphosphates, 0.42 μM Bovine Neospora Forward primerand 0.384 μM Bovine Neospora Reverse primer. After precycle denaturationat 94° C. for 4 min to reduce nonspecific amplification, 2.5 U of TaqDNA polymerase (Promega Corp., Madison, Wis.) was added and the mixturewas overlaid with 50 μl of mineral oil. Amplification was performed in aDNA Thermal Cycler (Perkin Elmer Cetus Corp., Norwalk, Conn.) for 31cycles as follows: denaturation at 94° C. for 1 min, annealing at 54° C.for 1 min, and extension at 72° C. for 2 min. The last cycle was given aprolonged extension period of 7 min. After amplification, 5 μl of eachsample or a BioMarker Low (BioVentures, Inc., Murfreeboro, Tenn.) DNAsize standard was mixed with 1 μl of 6× loading dye and electrophoresedon a 3% Nusieve 3:1 agarose gel (FMC Bioproducts). The gel was stainedin a 0.5 μg/ml ethidium bromide solution for 30 min and observed for thepresence of amplification products under ultraviolet illumination.

Oligonucleotide DNA Probes

3) BPA/Neospora Internal Probe Sequence (5'-AGTCAAACGCG-3'(SEQ ID NO: 7)

4) Toxoplasma Internal Probe Sequence (5'-AAGTCAACGCG-3'(SEQ ID NO: 8)

Amplification products were denatured in the gel and transferred tonylon membranes (Hybond-N; Amersham Corp., Arlington Heights, Ill.) bythe Southern blotting method; DNA was cross-linked to nylon membraneusing a Stratalinker UV crosslinker (Stratagene, La Jolla, Calif.).Prehybridization, preparation of the labeled internal probe, andhybridization were performed as recommended by the manufacturer for theEnhanced Chemiluminescence 3'-oligolabeling and Detection Systems(Amersham). Labeled internal probe was added to a final concentration of10 ng/ml of hybridization solution and incubated overnight at 30° C.with gentle agitation. After hybridization, the membranes were washedtwice for 5 min each at room temperature in 5× SSC and 0.1% (w/v) SDS,and then washed twice for 5 min each at room temperature in 0.5× SSC and0.1% (w/v) SDS. Membrane blocking, antibody incubations, signalgeneration and detection were performed as described by themanufacturer. Membranes were exposed to Kodak X-Omat film for 3-10 min.

Results

Using the Neospora-specific primers 294 bp PCR products were amplifiedfrom DNAs of BPA-1 and Toxoplasma (RH and BT isolates). In addition, a350 bp product was amplified from Sarcocystis cruzi DNA. No productswere produced with DNAs from various bacteria, CPAE cells, and bovinethymocytes. Only the Neospora-specific probe hybridized to the Neosporaamplification product. Similarly, the Toxoplasma-specific hybridizedonly to the Toxoplasma amplification product.

Example 5

This example describes experimental infections of pregnant cows withculture-derived Neospora tachyzoites.

Three cows were inoculated with 8×10⁶ tachyzoites of the BPA1 Neosporaisolate (3×10⁶ tachyzoites IV, and 5×10⁶ tachyzoites IM). These cowswere inoculated at 95 days gestation (Cow #412), 100 days gestation (Cow#416), and 105 days gestation (Cow #413). In each case, a Neospora fetalinfection was confirmed (Cow #412 expelled an infected mummified fetus;Cow #416 gave birth to a calf that was in utero exposed; and an infectedfetus was removed surgically from Cow #416). Two control cows wereinoculated with uninfected cell culture and gave birth to uninfectedlive calves.

These cows were kept and rebred without any intervention. All threeexperimental cows gave birth to seronegative, clinically normal calves(not all post mortem tissues examined to date).

The cows were kept and rebred once again. The previously infected cows(#s 412, 416, 413 were then rechallenged by giving them the sameinoculum (8×10⁶ tachyzoites, divided and given IV and IM) at 89, 83, and83 days gestation, respectively. Control cows were rebred and observed.Two infected cows (#s 413 and 416) gave birth to live calves which wereclinically normal and seronegative to Neospora antigens. The third cow(#412) aborted 27 days post inoculation. The fetus was recovered.Although mild lesions suggestive of Neospora infection were found,Neospora infection, to date, has not been confirmed (formalin-fixedparaffin embedded tissues negative by immunohistochemistry). The cow wasrebred and resorbed its fetus. She was rebred again and she aborted onceagain at 97 days gestation. This second fetus was not recovered. Thusfar histopathologic examination or the tissues from the two clinicallynormal calves indicates that they were not tranplacentally infected withNeospora parasites.

This is the first experiment to show that cattle can be protectedagainst Neospora abortion by immunization with culture-derivedtachyzoites of the BPA-1 Neospora isolate.

Example 6

This example describes identification of two clones from Neospora cDNAlibrary. These cDNAs can be used to produce recombinant immunodominantproteins useful for vaccines and immunodiagnostics.

Methods and Methods

In vitro cultivation

Neospora tachyzoites (BPA-1 isolate) and Toxoplasma gondii (RH strain)tachyzoites were cultivated in tissue culture as described in Conrad,Parasitology, 106:239-249 (1993). Briefly, BPA-1 tachzoites were grownin confluent layers of BAE (bovine aortic endothelial) cells, harvestedand filtered through 5 μm disc filters to remove cellular debris, washedtwice in phosphate buffered saline (PBS), and pelleted for use. AftermRNA for cDNA library construction, BPA-1 and Toxoplasma tachyzoiteswere subsequently grown in Vero cells.

Isolation of nucleic acids

Total RNA was isolated from pelleted tissue culture cells with TRISOLV™reagent (Biofecx Laboratories, Houston, Tex.). Poly A⁺ RNA was selectedby oligo (dT)-cellulose chromatography (Avis, et al., Proc. Natl. Acad.Sci. USA 69, 1408-1412 (1972)). Integrity of RNA was monitored byelectrophoresis on a formaldehyde gel and visualized with ethidiumbromide using standard methods.

DNA was prepared with the IsoQuick NDA Extraction Kit (Microprobe Corp.,Garden Grove, Calif.).

Construction of a bovine Neospora cDNA library in λgt11

Construction of a Neospora cDNA expression library in the vector λgt11was performed essentially according to the manufacturer's protocol inthe cDNA Synthesis Kit (Stratagene, La Jolla, Calif.). In short, firststrand cDNA was synthesized from 5 μg of tachyzoitic poly(A) RNA usingStrataScript R Nase H reverse transcriptase and the second strand wassynthesized with E. coli DNA polymerase. Subsequently, the doublestranded cDNA was blunt-ended with the Klenow fragment, ligated to EcoR1 adaptors on both ends, kinased with T4 polynucleotide, andsize-fractionated on a Sephacryl S-400 spin column. The final cDNAproduct was then ligated into the Eco R1 digested and dephosphorylatedλgt11 vector and packaged using the Gigapack III Gold Packaging Extract(Stratagene, La Jolla, Calif.). The library contained 7×10⁶ phages withapproximately 97% of these being recombinants. Subsequently, one roundof library amplification was completed in E. coli Y1088.

Immunoscreening of the cDNA library

The library was plated on E. coli Y1090r- and duplicate nitrocelluloseplate lifts were screened with high titer sera from naturally infectedcow D91-4696 and experimentally infected cow #416 described in Conrad,et al., J. Vet. Diagn. Invest., 5:572-578 (1993). Sera were firstpre-absorbed with Y1090r-bacterial lysates and then diluted 1:300 inTBS-T (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.05% Tween 20) containing 5%horse serum and used for screening. Bound antibodies were visualizedwith alkaline phosphatase conjugated goat anti-bovine IgG diluted 1:5000in TBS-T, 5% HS. The immunoscreening procedure was essentially asdescribed for immunoblot assays below except 5% nonfat dry milk wasreplaced with 5% horse serum.

DNA Sequencing

PCR products generated with universal λgt11 primers flanking thevector's Eco R1 restriction site (Promega, Madison, Wis.) were used astemplates for sequencing. Cycling conditions were as described by Obar,et al., Methods in Cell Biology, 37, 361-406 (1993). Two sets oftemplates for sequencing were made for each clone and both weresequenced in the forward and reverse directions.

Automated DNA sequencing was accomplished with the ABI 373 DNA Sequencerand the DNA Sequencing Kit with AmphiTaq Polymerase FS (Perkin-ElmerCorp., Foster City, Calif.). Dye terminator chemistry in conjunctionwith cycle sequencing was used.

Southern and Northern blot analysis

For Southern blotting, 4 ug of genomic DNA was digested with restrictionenzyme and separated on a 0.8% agarose gel according to standardprocedures. After the gel was depurinated with 250 mM HCl for 10minutes, denatured with 1.5M NaCl, 0.5M Tris-HCl, pH 7.5 for 30 minutes,the nucleic acids were transferred overnight to Hybond-Nnylon membranes(Amersham Corp., Arlington Heights, Ill.) and crosslinked to themembrane via a UV crosslinker (Stratagene, La Jolla, Calif.). Forprobes, Eco R1 inserts from the recombinant clones were labeled with theECI Direct Nucleic Acid Labeling and Detection Systems (Amersham Corp.).Blots were prehybridized with the ECL Gold Hybridization buffercontaining 5% blocking agent and 0.5M NaCl (Amersham Corp.) for 1 hourand hybridized with the probes overnight at 42° C. The blots were washedat 42° C. (2×20 min.) in 6M Urea, 0.4% SDS, 0.5× SSC and rinsed with 20×SSC prior to autoradiography.

For Northern blotting, the above Southern blot procedure was essentiallyfollowed except that 0.4 ug mRNA (clone N54) or 5 ug of total RNA (cloneN57) were run on a 1% formaldehyde gel and no extensive treatment of thegel was required prior to transfer. Also, when the Eco R1 insert fromclone N54 was used as a probe, the wash protocol was adjusted to 0.4%SDS, 0.5× SSC at 45° C. (2×20 min.).

Recombinant Protein Expression and Purification

To construct the expression vector, PCR products of the clone insertswere digested with Eco R1 and gel purified. This DNA was ligated intothe Eco R1 digested and dephosphorylated pRSET B vector (Kroll, et al.,DNA and Cell Biology, 12 441-453 (1993)) and the resulting plasmid wastransformed into BL21 DE3 pLysS E. coli (Studier, et al., MethodsEnzymol. 185, 60-89 (1990)). Fusion proteins expressed from this plasmidcontained a hexamer of histidines which allowed purification through aNi²⁺ affinity column under denaturing conditions.

Single colonies with high expression levels were inoculated into LuriaBroth containing 100 ug/mL ampicillin and grown at 37° C. to an O.D.₆₀₀of 0.6. Overexpression of fusion proteins was initiated with theaddition of isopropyl-B D-thiogalactopyranoside (0.4 mM final) andshaking for 3 hours. Affinity purification with a nickel-NTA-agaroseaffinity column (Qiagen, Chetsworth, Calif.) under denaturing conditionswas completed as described by Kroll, et al., DNA and Cell Biology, 12441-453 (1993). Samples were concentrated with a Centricon-10 (Amicon,Beverly, Mass.) and further purified by (size chromatography) withSephadex G-150 superfine (Pharmacia, Uppsala, Sweden) equilibrated in 8Murea, 0.1M Naphosphate, 0.01M Tris, pH 4.5 using standard methods.Denaturants in the final proteins were removed by a slow 1M ureastepwise dialysis to a final PBS, pH 7.4 buffer. Samples wereconcentrated again with Centricon-10 (Amicon, Beverly, Mass.) andquantitated with the BCA Protein Assay (Pierce, Rockford, Ill.).

Polyclonal Antibody Production

Female New Zealand White rabbits were immunized subcutaneously with 400ug of recombinant proteins in a 50% emulsion of Freund's completeadjuvant and thereafter boosted twice at 4 week intervals with 200 ugand then 100 ug of protein in incomplete Freund's adjuvant. Sera wereobtained two weeks after the third immunization and used forimmunoblots.

Immunoblots

Proteins were analyzed on a 12% polyacrylamide gel or a 4-20% gradientslab gel (Anderson, et al. J. Am. Vet. Med. Assoc., 207:1206-1210(1995)). Parasite antigens were harvested from tissue culture asdescribed above, washed 2× in PBS, lysed with water, freeze thawed 3×,and sonicated. Both recombinant antigens and parasite antigens werequantitated with the BCA Protein Assay (Pierce), denatured in Laemmli'ssample buffer and boiled for 5 minutes prior to electrophoresis.SDS-PAGE and Western blots were performed under standard conditions.

Rabbit antisera to the recombinant antigens, Neospora (BPA1) (Conrad,Parasitology, 106:239-249 (1993)), and Toxoplasma gondii (Conrad,Parasitology, 106:239-249 (1993)) were diluted 1:300 in TBS-Tween, 5%Blotto and incubated for 5 hours at room temperature. The secondaryantibody, HRP-goat anti-rabbit (Jackson Laboratories) was diluted 1:1000in the same buffer and incubated for 2 hours at room temperature. Blotswere developed with 4-chloronaphthol and H₂ O₂.

Results

After one round of amplification, a bovine Neospora λgt11 cDNA librarywas generated containing 5.3×10¹⁰ pfu/mL. Sera from cow D91-4696 and cow416 which had high titers to Neospora antigens were chosen to screen thelibrary. No significant banding pattern differences were seen betweenWestern blots which were incubated with these sera (data not shown).Both naturally infected cow D91-4696 and experimentally infected cow 416tested negative for Toxoplasma gondii. Experimentally infected cow 416was a subject of a previous Neospora abortion study (Conrad, et al., J.Vet. Diagn. Invest., 5:572-578 (1993)).

Primary screening of 200,000 cDNA clones with the above sera identified61 double positive clones. Two were further characterized and designatedN54 and N57. Based on agarose gel electrophoresis, the λgt11 Eco R1inserts were 430 and 630 base pairs respectively.

Sequence analysis of clone N54 revealed that the cDNA insert was 407bases within an open reading frame. The clone was not full length asindicated by the absence of a 5'-methione start site and the absence ofa 3' stop codon and poly A tail (SEQ ID NO: 9). Translation of thesequence into its corresponding amino acids revealed that the proteinsequence was highly proline rich (32%) and composed of several repeatunits (SEQ ID NO: 10). Of particular interest is the sequence,SPPQS(S/Y)PPEP(SEQ ID NO: 13) shown in bold in FIG. 5A, which occurstwice and has a high surface probability (Emini ave. index=3.025) andmoderate antigenicity index (Jameson-Wolf ave. index=1.24). Other uniquerepeats within the region are the tetrapeptides, HP(H/P)P(SEQ ID NO: 14)and SPP(E/Q)(SEQ ID NO: 15), which occur four times each within the 135amino acid sequence and the sequence, SY(AIP)P(D/E)PSP, which containsconserved substitutions.

Unlike clone N54, clone N57 contained a 3' stop codon and a long poly Atail (SEQ ID NO: 11); however, because of difficulties in sequencing therepetitive noncoding region, only the coding sequences are shown (SEQ IDNO: 12). While this partial clone did not have any discernible peptiderepeats within its 76 amino acid sequence, upon inspection at the DNAlevel, multiple units of nucleotide repeats were identified. Inparticular, a long tandem repeat with 74% homology was present at the 3'end (FIG. 5B). While these repeats were similar at the nucleotide level,the multiple deletions within the repeats resulted in frame shifts thattranslated into peptide sequences with only 2 out of 10 amino acidsbeing similar. By Emini analysis, these two regions appeared to belikely surface exposed and a correlation between surface exposure andantigenicity was seen. The second repeat also appeared to have apotential glycosylation at the position 50 asparagine. In addition,there was a high frequency of nine GGA(A/G) repeats clustered within a105 base region at the 3' end of the gene sequence.

Clones N54 and N57 hybridized to bands of different molecular weights onSouthern blots of Neospora (BPA-1) DNA, indicating that the clones werederived from separate Neospora genes. Clone N54 showed somehybridization to a higher molecular weight band of Toxoplasma gondiiDNA, whereas clone N57 did not bind to Toxoplasma DNA or to Vero DNA.

Northern blots with the same probes also showed that both clonesrecognized different molecular weight RNA transcripts and thus, encodeddistinct Neospora proteins. Clone N54 bound to a 4.2 kb Neosporatranscript, while clone N57 bound to a shorter 1.4 kb transcript.Neither clones hybridized to Toxoplasma gondii RNA or Vero RNA.

When clones N54 and N57 were expressed as histidine fusion proteins fromthe pRSET vectors, the resulting protein product of clone N54 was 29.3kD whereas the protine product of clone N57 was 20.1 kD. By Western blotanalysis, both proteins were recognized by rabbit antisera to BPA1 andnot by rabbit Toxoplasma gondii antisera (data not shown). This suggeststhat the recombinant antigens are more diagnostically useful than thewhole lysate which did have some reactivity to rabbit Toxoplasma gondiiantisera.

In addition, polyclonal monospecific antisera to clones N54 and N57 onlybound to Neospora antigens on reducing and nonreducing Western blots.Rabbit anti-N54 recognized Neospora bands of molecular weights 97.2 kD,87.9 kD, 77.1 kD, 67.4 kD, 64.3 kD, 60.1 kD, 55.3 kD, and 28.3-28(reducing) and 126.7 kD, 89.4 kD, 68.7 kD, 58.4 kD), 55.2 kD, 54.5 kD,52.7 kD, 49.7 kD, 46.7 kD and 26.5-27.9 (nonreducing). Rabbit anti-N57recognized Neospora bands to molecular weights 33.6 kD, 31.4 kD, 27.5kD, and 22.4 kD (reducing) and 32.8 kD, 30.6 kD, 28.4 kD, 26.3 kD, and21.0 kD (nonreducing). Neither polyclonal antisera bound to Toxoplasmagondii antigens nor to Vero cell antigens, making these two recombinantantigens promising candidates for a highly specific ELISA.

Conclusion

Current ELISA protocols require the use of in vitro cultivated ofNeospora tachyzoites for coating antigens. (Pare, et al., J. Vet. Diag.Invest., 7:352-9 (1995); Bjorkman, et al., Parasite Immunology,16:643-8). When using such a crude mixture of antigens, one runs therisk of generating false positives due to crossreactivity betweenproteins from closely related parasites. By using one or two Neosporaspecific recombinant antigens in an ELISA, potentially crossreactiveantigens are removed. In addition, the use of whole parasites requirestime consuming and expensive tissue culture methods.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 17    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1747 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AGTCATATGCTTGTCTTAAAGATTAAGCCATGCATGTCTAAGTATAAGCTTTTATACGGC60    TAAACTGCGAATGGCTCATTAAAACAGTTATAGTTTATTTGATGGTCTTTACTACATGGA120    TAACCGTGGTAATTCTATGGCTAATACATGCGCACATGCCTCTTCCTCTGGAAGGGCAGT180    GTTTATTAGATACAGAACCAACCCACCTTCCGGTGGTCCTCGGGTGATTCATAGTAACCG240    AACGGATCGCGTTTGACTTCGGTCTGCGACGGATCATTCAAGTTTCTGACCTATCAGCTT300    TCGACGGTACTGTATTGGACTACCGTGGCAGTGACGGGTAACGGGGAATTAGGGTTCGAT360    TCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAAT420    TACCCAATCCTGATTCAGGGAGGTAGTGACAAGAAATAACAACACTGGAAATTTCATTTC480    TAGTGATTGGAATGATAGGAATCCAAACCCCTTTCAGAGTAACAATTGGAGGGCAAGTCT540    GGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAA600    AAAGCTCGTAGTTGGATTTCTGCTGGAAGCAGCCAGTCCGCCCTCAGGGGTGTGCACTTG660    GTGAATTCTAGCATCCTTCTGGATTTCTTCACACTTCATTGTGTGGAGTTTTTTCCAGGA720    CTTTTACTTTGAGAAAATTAGAGTGTTTCAAGCAGGCTTGTCGCCTTGAATACTGCAGCA780    TGGAATAATAAGATAGGATTTCGGCCCTATTTTGTTGGTTTCTAGGACTGAAGTAATGAT840    TAATAGGGACGGTTGGGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTAGATTTGT900    TAAAGACGAACTACTGCGAAAGCATTTGCCAAAGATGTTTTCATTAATCAAGAACGAAAG960    TTAGGGGCTCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAG1020    AGATAGGAAAACGTCATGCTTGACTTCTCCTGCACCTTATGAGAAATCAAAGTCTTTGGG1080    TTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC1140    AGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAT1200    AGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGT1260    TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGC1320    TAAATAGGATCAGGAACTTCGTGTTCTTGTATCACTTCTTAGAGGGACTTTGCGTGTCTA1380    ACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCAC1440    GCGCGCTACACTGATGCATCCAACGAGTTTATAACCTTGGCCGATAGGTCTAGGTAATCT1500    TGTGAGTATGCATCGTGATGGGGATAGATTATTGCAATTATTAATCTTCAACGAGGAATG1560    CCTAGTAGGCGCAAGTCAGCAGCTTGCGCCGATTACGTCCCTGCCCTTTGTACACACCGC1620    CCGTCGCTCCTACCGATTGAGTGTTCCGGTGAATTATTCGGACCGTTTTGTGGCGCGTTC1680    GTGCCCGAAATGGGAAGTTTTGTGAACCTTAACACTTAGAGGAAGGAGAAGTCGTAACAA1740    GGTTTCC1747    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 52 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CCGAATTCGTCGACACCTGGTTGATCCCCGACGACCGTGGTCTGAACGGGAG52    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGGCCCTAGGTGGCGCCGACGACCGTGGTCTGAACGGGAG40    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTAC39    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    AAGTATAAGCTTTTATACGGCT22    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CACTGCCACGGTAGTCCAATAC22    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AGTCAAACGCG11    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    AAGTCAACGCG11    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 407 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 2..406    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GCAACATCCTCATCCTCATCCTCCTCGCCCTAATCCCCCCGAGGCT46    GlnHisProHisProHisProProArgProAsnProProGluAla    151015    TCACCTCCGCAGTCGTCTCCGCCCGAACCTCAACGTCCTTTTTCTCAG94    SerProProGlnSerSerProProGluProGlnArgProPheSerGln    202530    TGGCCCCACACTCCACATTTTTTTCATTATCATCCCTACCCTGGATAT142    TrpProHisThrProHisPhePheHisTyrHisProTyrProGlyTyr    354045    AATCTTCCTTATTTTACTTACCATCAGTCTCCTCTTCCTTATGGTCCC190    AsnLeuProTyrPheThrTyrHisGlnSerProLeuProTyrGlyPro    505560    TACGGAAGGGATCCTTGTCCTTGTGCTTCCCACCCTTATCCCGCCGAC238    TyrGlyArgAspProCysProCysAlaSerHisProTyrProAlaAsp    657075    GATTCACCTCTGGGGTCGTATGCGCCCGATCCATCACCTCCCCAGTCT286    AspSerProLeuGlySerTyrAlaProAspProSerProProGlnSer    80859095    TATCCCCCAGAACCTTCACCATCGAAGCCGTCTCCACCAGAGGGTTCT334    TyrProProGluProSerProSerLysProSerProProGluGlySer    100105110    TCTCCGAGAGTCCCTTCGCCTCACCGGCATCCCAGCAGATCACGTCTC382    SerProArgValProSerProHisArgHisProSerArgSerArgLeu    115120125    CCCTCTGCTGTCGAGCCATCTCCAC407    ProSerAlaValGluProSerPro    130135    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 135 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GlnHisProHisProHisProProArgProAsnProProGluAlaSer    151015    ProProGlnSerSerProProGluProGlnArgProPheSerGlnTrp    202530    ProHisThrProHisPhePheHisTyrHisProTyrProGlyTyrAsn    354045    LeuProTyrPheThrTyrHisGlnSerProLeuProTyrGlyProTyr    505560    GlyArgAspProCysProCysAlaSerHisProTyrProAlaAspAsp    65707580    SerProLeuGlySerTyrAlaProAspProSerProProGlnSerTyr    859095    ProProGluProSerProSerLysProSerProProGluGlySerSer    100105110    ProArgValProSerProHisArgHisProSerArgSerArgLeuPro    115120125    SerAlaValGluProSerPro    130135    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 232 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 2..229    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CATAGCCGCGTTAGTTGCAGCAGCCGCATTTGCTGGATTGGGTCTC46    IleAlaAlaLeuValAlaAlaAlaAlaPheAlaGlyLeuGlyLeu    151015    GCGAGAACATTCAGGCATTTCGTGCCAAAAAAGTCAAAGACGGTTGCG94    AlaArgThrPheArgHisPheValProLysLysSerLysThrValAla    202530    AGTGAGGACTCTGCGCTCGGAAACAGTGAAGAGCAGTATGTGGAAGGA142    SerGluAspSerAlaLeuGlyAsnSerGluGluGlnTyrValGluGly    354045    ACCGTGAACGGGAGCAGTGATCCGGAACAGGAGCGGGCGGGTGGGCCT190    ThrValAsnGlySerSerAspProGluGlnGluArgAlaGlyGlyPro    505560    CTTATCCCGGAAGGAGACGAGCAGGAAGTAGACACCGAATAG232    LeuIleProGluGlyAspGluGlnGluValAspThrGlu    657075    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 76 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    IleAlaAlaLeuValAlaAlaAlaAlaPheAlaGlyLeuGlyLeuAla    151015    ArgThrPheArgHisPheValProLysLysSerLysThrValAlaSer    202530    GluAspSerAlaLeuGlyAsnSerGluGluGlnTyrValGluGlyThr    354045    ValAsnGlySerSerAspProGluGlnGluArgAlaGlyGlyProLeu    505560    IleProGluGlyAspGluGlnGluValAspThrGlu    657075    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 6    (D) OTHER INFORMATION: /product="OTHER"    /note= "Xaa = Ser or Tyr"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    SerProProGlnSerXaaProProGluPro    1510    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3    (D) OTHER INFORMATION: /product="OTHER"    /note= "Xaa = His or Pro"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    HisProXaaPro    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    SerProProGlx    1    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 57 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GGAAACAGTGAAGAGCAGTATGTGGAAGGAACCGTGAACGGGAGCAGTGATCCGGAA57    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3    (D) OTHER INFORMATION: /product="OTHER"    /note= "Xaa = Ala or Pro"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 5    (D) OTHER INFORMATION: /product="OTHER"    /note= "Xaa = Asp or Glu"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    SerTyrXaaProXaaProSer    15    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide which encodes a polypeptide having a sequence as shown in SEQ ID NO:
 10. 2. The polynucleotide of claim 1, wherein the polynucleotide has a sequence as shown in SEQ ID NO:
 9. 3. An isolated polynucleotide which encodes a polypeptide having a sequence as shown in SEQ ID NO:
 12. 4. The polynucleotide of claim 3, wherein the polynucleotide has a sequence as shown in SEQ ID NO:
 11. 